Condensin phosphorylated by the Aurora-B-like kinase Ark1 is ...

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Research Article

Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2 Norihiko Nakazawa1, Rajesh Mehrotra2,*, Masahiro Ebe2 and Mitsuhiro Yanagida1,2,‡ 1

Okinawa Institute and Science Technology Promotion Corporation, 1919-1 Tancha, Onna-son, Kunigami, Okinawa 904-0412, Japan CREST Research Program, Japan Science and Technology Corporation, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan 2

*Present address: Department of Biological Sciences, BITS, Pilani, Rajasthan 333031, India ‡ Author for correspondence ([email protected])

Journal of Cell Science

Accepted 13 January 2011 Journal of Cell Science 124, 1795-1807 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.078733

Summary Condensin is a conserved protein complex that functions in chromosome condensation and segregation. It has not been previously unequivocally determined whether condensin is required throughout mitosis. Here, we examined whether Schizosaccharomyces pombe condensin continuously acts on chromosomes during mitosis and compared its role with that of DNA topoisomerase II (Top2). Using double mutants containing a temperature-sensitive allele of the condensin SMC2 subunit cut14 (cut14-208) or of top2, together with the cold-sensitive nda3-KM311 mutation (in -tubulin), temperature-shift experiments were performed. These experiments allowed inactivation of condensin or Top2 at various stages throughout mitosis, even after late anaphase. The results established that mitotic chromosomes require condensin and Top2 throughout mitosis, even in telophase. We then showed that the Cnd2 subunit of condensin (also known as Barren) is the target subunit of Aurora-B-like kinase Ark1 and that Ark1-mediated phosphorylation of Cnd2 occurred throughout mitosis. The phosphorylation sites in Cnd2 were determined by mass spectrometry, and alanine and glutamate residue replacement mutant constructs for these sites were constructed. Alanine substitution mutants of Cnd2, which mimic the unphosphorylated protein, exhibited broad mitotic defects, including at telophase, and overexpression of these constructs caused a severe dominantnegative effect. By contrast, glutamate substitution mutants, which mimic the phosphorylated protein, alleviated the segregation defect in Ark1-inhibited cells. In telophase, the condensin subunits in cut14-208 mutant accumulated in lumps that contained telomeric DNA and proteins that failed to segregate. Condensin might thus serve to keep the segregated chromosomes apart during telophase. Key words: Condensation, Segregation, Condensin, Aurora kinase, Top2

Introduction Mitotic chromosome condensation is an important event that is a prerequisite for accurate chromosome segregation in anaphase (Koshland and Strunnikov, 1996; McHugh and Heck, 2003; Swedlow and Hirano, 2003; Yanagida, 2005). Although mitotic chromosome condensation has been well described overall, the precise details of the relevant molecular mechanisms have remained enigmatic (Belmont, 2006; Gassmann et al., 2004). Even in unicellular eukaryotic organisms, such as yeast, interphase chromatin DNA is already compacted ~1000-fold in the nucleus, relative to the length of the naked DNA duplex. Forming properly condensed chromosomes upon entry into mitosis at prophase requires a further compaction, by severalfold, of interphase chromatin DNA (Bak et al., 1977; Earnshaw, 1988; Kireeva et al., 2004). In the fission yeast Schizosaccharomyces pombe, which has a small genome, chromatin condenses ~fivefold upon mitotic entry, judging from fluorescence in situ hybridization (FISH) images of chromosome arms (Saka et al., 1994). This is a marginal degree of mitotic condensation, but it is absolutely essential for proper segregation. The condensation might be necessary to promote the final compaction and resolve sister chromatids by metaphase and produce a chromosomal architecture that can endure segregation (e.g. against the pulling force of the mitotic spindle). Another possibility is that chromosome condensation serves to remove the

many proteins and RNAs that bind to interphase chromosomes and that could interfere with segregation (Yanagida, 2009). These two views are not mutually exclusive. The evolutionarily conserved condensin complex plays a fundamental role in mitotic chromosome condensation (Hirano, 2005; Hudson et al., 2009). This complex has DNA-dependent ATPase activity, which promotes positive DNA supercoiling (Kimura and Hirano, 1997). ATPase motifs are present in the two ‘structural maintenance of chromosomes’ (SMC) (Strunnikov et al., 1993) subunits (Cut3 and Cut14), which also have coiled-coil and hinge regions; three non-SMC condensin (Cnd1, Cnd2 and Cnd3) subunits are bound to the ATPase-containing globular domains of the SMC heterodimer (Yoshimura et al., 2002) (see Fig. 5A). The mitotic functions of condensin seem to be mediated by its phosphorylation in mitosis. The kinase Cdc2 activates the DNA supercoiling activity of the Xenopus condensin holocomplex in vitro (Kimura et al., 1998). In fission yeast, the N-terminal T19 residue of the SMC condensin subunit Cut3 (also known as SMC4) is phosphorylated by mitotic kinase Cdc2, and an unphosphorylatable mutant causes a severe defect in mitotic condensation owing to the retention of condensin in the cytoplasm (Nakazawa et al., 2008; Sutani et al., 1999). The requirement of kinase Aurora B for condensin recruitment to mitotic chromosomes was initially reported in Drosophila (Giet and Glover, 2001).


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Journal of Cell Science 124 (11)

Studies in budding yeast have also shown that mitotic chromosome condensation requires a functional Aurora B kinase (Lavoie et al., 2004). Studies of vertebrate condensin suggested that Aurora B contributes to the efficient localization of condensin I (Lipp et al., 2007; Takemoto et al., 2007) and/or to the accumulation of condensin II at the kinetochore (Ono et al., 2004). In fission yeast, Cdc2 phosphorylation is not sufficient for docking condensin to mitotic chromatin, and phosphorylation by both Cdc2 and Ark1 (the fission yeast Aurora B homologue) is necessary (Nakazawa et al., 2008). An essential role of the polo kinase in ensuring anaphasespecific condensation has also been recently shown in budding yeast (St-Pierre et al., 2009). Evidence for the essential role of condensin in mitotic chromosome condensation and subsequent segregation has been obtained by inactivating one of the condensin subunits before mitotic entry with the use of temperature-sensitive (ts) mutations, transposon insertion, conditional shut-off and antibody or RNA interference (RNAi) knockdown (Bhat et al., 1996; Hagstrom et al., 2002; Hirano and Mitchison, 1994; Hudson et al., 2003; Ono et al., 2004; Saka et al., 1994; Strunnikov et al., 1995). A study to examine the role of condensin after metaphase was performed by adding antibodies against condensin to Xenopus in vitro extracts immediately after the addition of Ca2+, for the release from metaphase, and a chromosome non-disjunction phenotype was observed (Wignall et al., 2003). Similarly, antibody blocking of condensin was performed in frog egg extracts after condensation, and a resulting deformation of mitotic chromosomes found (Hirano and Mitchison, 1994). In vivo, the fate of the mitotic chromosome was examined after condensation in the presence of nocodazole, using budding yeast temperature-sensitive condensin mutants; after the shift to the restrictive temperature, the amount of condensed rDNA was substantially diminished, suggesting that condensin is required after condensation (Lavoie et al., 2002). Nevertheless, to our knowledge, no attempt has been made to examine the role of condensin after chromosome segregation. In this study, we investigated the effect of condensin inactivation through two approaches. The first approach was to employ a specific double mutant that is suitable for examining the continuing role of condensin in mitosis. This mutant contains the cold-sensitive (cs) -tubulin nda3-KM311 mutation and the temperature-sensitive cut14-208 mutation, and it was used for one- and two-step temperature shift-up experiments. Our second approach was to analyze the phosphorylation sites of condensin subunits during mitosis. We identified ~18 mitotic phosphorylation sites by mass spectrometry and focused on the role of sites phosphorylated by the Aurora-B-like kinase Ark1. These sites included the phosphorylated S52 residue in the non-SMC Cnd2 subunit (also known as Barren). Phosphorylation-specific antibodies directed against this residue showed that the intense mitotic phosphorylation of Cnd2 was dependent on the presence of Ark1. We constructed several alanine substitution cnd2 mutants, whose severe defects suggested a continuous role for phosphorylated condensin during mitosis. As DNA topoisomerase II (Top2) is also required for both condensation and segregation (Uemura et al., 1987), we also examined in detail the phenotypes of the nda3-cs top2-ts double mutant and compared these phenotypes with those of the nda3-cs cut14-ts mutant. Our results strongly suggest that condensin and Top2 are both required throughout mitosis for proper chromosome maintenance, although they might influence distinct chromosomal areas.

Results Inactivation of condensin after mitotic entry causes missegregation

We first addressed whether S. pombe condensin is required after chromosome condensation in mitosis. Previously, defects in condensation and segregation have been observed when S. pombe temperature-sensitive condensin mutants comprising largely G2 phase cells were transferred into restrictive conditions (Aono et al., 2002; Saka et al., 1994). It was thus of interest to inactivate condensin after mitotic entry to examine its immediate effect on segregation. For this purpose, the temperature-sensitive mutant cut14-208 (Saka et al., 1994) was employed. The cut14-208 mutant protein, which contains the mutation S861P (Sutani and Yanagida, 1997) in the coiled-coil region, remains functional at permissive temperatures (26–30°C) but is quickly inactivated by a change of temperature to 36°C (restrictive temperature). The cut14-208 mutant was crossed with the cold-sensitive -tubulin mutant nda3-KM311 (Hiraoka et al., 1984) to obtain the nda3-cs cut14-ts double mutant. The Nda3-KM311 protein fails to assemble microtubules at 20– 22°C but is rapidly reactivated by a shift to 30–36°C. Thus, the double-mutant strain produces colonies at 30°C but not at 22°C or 36°C (Fig. 1A). A series of results (Fig. 1B–F) established that condensin has to be functional even after chromosome condensation. The doublemutant cells arrested after entry into mitosis at 20°C: they contained condensed chromosomes but, owing to the absence of assembled microtubules, were unable to proceed further. At 36°C (the permissive temperature for nda3), however, the double-mutant cells were released for mitotic progression following the reactivation of tubulin, whereas the condensin mutant protein Cut14-208 was inactive. The control single-mutant nda3-cs and the double-mutant nda3-cs cut14-ts strains were first grown at 30°C and then shifted to 20°C for 8 hours (Fig. 1B, Nda3– Cut14+). Both strains arrested at an early mitotic stage, revealing condensed chromosomes (Fig. 1C) [DNA was stained with the fluorescent probe 4,6-diamino-2-phenylindole (DAPI)]. The cultures were then shifted to 36°C (Fig. 1B, Nda3+ Cut14– for the double mutant) and aliquots were taken at intervals for 60 minutes. After 10 minutes (corresponding to early anaphase) and 15 minutes (corresponding to late anaphase), the double-mutant cells where condensin was inactivated post-condensation showed frequent abnormal segregation, whereas normal segregation was observed for the control single-mutant nda3 strain (Fig. 1D); ~50% of the doublemutant cells displayed highly streaked or extended chromosomal DNA (Fig. 1D, arrows). Quantitative results (Fig. 1E) showed that the percentage of early mitotic cells (designated condensed) of the control single nda3 mutant (left-hand panel) decreased to the minimal level after 10 minutes, whereas the percentage of cells with partially separated chromosomes peaked at 40% at 5 minutes after the temperature shift. Normal chromosome segregation (two nuclei) had occurred in 60–65% of the cells at 10–15 minutes. In the double mutant with inactive condensin, the percentage of cells with partially segregated chromosomes also peaked at 40% at 5 minutes after the temperature shift, but chromosomes in less than 20% of the cells had segregated into the two daughter nuclei at 10–15 minutes (right-hand panel). The percentage of cells with the aberrant extended chromosomes instead peaked at 55% at ~10–20 minutes after the shift. The segregating daughter nuclei observed in the double mutant were frequently abnormal and differently sized, and they displayed trailing chromosomes in the middle of the cell. Thus, condensin


Role of condensin throughout mitosis

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Fig. 1. Condensin Cut14 is required after condensation. (A)The single nda3 and cut14 mutants and the double mutant produce colonies at 30°C. WT, wild type. (B)A schematic of the temperature shift-up experiment using the double mutant. (C)The single nda3 mutant and double-mutant cells arrested in mitosis stained with DAPI. (D)Mitotically arrested cells were released for 10 and 15 minutes by a shift to 36°C and stained with DAPI. The arrows indicate abnormally extended chromosomes. (E)The percentage of cells with the indicated phenotypes observed in the single nda3 (left-hand panel) and the double-mutant nda3 cut14 cells (right-hand panel). (F)Time-lapse micrographs of the temperature-shift experiment depicted at the top. See the captions of the supplementary material Movies 1 and 2 for the detailed protocol. Chromatin DNA was visualized using a gene encoding RFP-tagged histone H2A under the control of the native promoter. The numbers in the panels indicate time in minutes. Scale bars: 10mm.

inactivation in the post-condensation period causes an aneuploidylike situation or the physical failure in chromosome segregation. Movies of living cells were taken with a DeltaVision microscope. The histone H2A gene (hta1+) tagged with red fluorescent protein (RFP) was chromosomally integrated under its native promoter in the single nda3-cs and the double nda3-cs cut14-ts mutants. Timelapse micrographs of representative cells are shown in Fig. 1F (supplementary material Movies 1 and 2). In the control nda3 mutant cell, chromosomes had separated normally at 6–10 minutes after the release to 36°C. For the condensin-inactivated double mutant (right-hand panels), aberrant chromosomes (streaked in cell 1 and decondensed in cell 2) were observed as soon as 3 minutes after the shift. The ‘streaking’ segregation, as seen in cell 1, was more frequent than the decondensation abnormality seen in cell 2. Thus, the inactivation of Cut14 and the resulting abnormal chromosome segregation occur very rapidly, at the order of 80% of the lumps (Fig. 4H, top panels). FISH signals produced by the telomere probe cos212 (Funabiki et al., 1993), which contains the telomereadjacent DNA of chromosomes I and II, were also frequently observed at the lumps (Fig. 4H, bottom panels). Thus, condensin inactivation in late anaphase caused the missegregation of chromosome termini bound to mutant condensin. Whether DNA regions other than telomere-adjacent sequences are present in the lumps remains to be determined. Condensin subunits are mitotically phosphorylated

To investigate how the post-transcriptional modification of condensin affects the mode of chromosome segregation, we next attempted to identify phosphorylation sites in mitotic condensin by mass spectrometry. Mitotic cells were obtained by employing the tubulin mutant nda3-KM311, which was chromosomally integrated with a FLAG-tagged wild-type cut14+ gene (Cut14-3FLAG) under its native promoter (Nakazawa et al., 2008). Large-scale cultures of mitotic cells (5⫻1010 cells) were grown at 20°C for 8 hours, extracts were prepared and anti-FLAG antibodies were used to immunoprecipitate condensin holocomplexes. The precipitates



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Fig. 4. The Cut14 mutant protein is present in a lump at late anaphase that also contains DNA. (A,B)GFP-tagged Cnd1, a non-SMC condensin subunit, was observed in the single nda3 mutant (A) and the double nda3-cs cut14-ts mutant (B) at 20°C, 30°C and 36°C (the indicated times are after the shift-up). Cells were fixed with methanol. Cnd1–GFP was present in the lump, which also contained DNA in the double mutant (indicated by the arrow). The frequencies of cells with signals that colocalized with DNA are indicated in parentheses on the right-hand side. (C–F) The lump was observed in the nda3 cut14 double mutant (D,F) when Cut3–GFP and Cnd2–GFP were used. (G)The nda3 cut14 double-mutant cells expressing CENP-A–GFP (SpCENP-A-GFP) were observed after the two-step shift-up (top panel). The double-mutant cells, after the two-step temperature shift, were observed after pericentromeric (Cen) FISH analysis (bottom panel). (H)Double-mutant cells with an integrated Taz1–GFP gene were observed after the two-step shift-up (top panel). Double-mutant cells after the two-step shift-up were observed after FISH with the telomere-adjacent sequence probe (cos212) (bottom panel). The percentages of cells with signals that colocalized with DNA are indicated in parentheses on the right-hand side for G and H. Scale bars: 10mm.

contained the five authentic subunits (Sakai et al., 2003; Sutani et al., 1999) (Fig. 5A), as compared with the silver-staining pattern of condensin isolated with the untagged control (Fig. 5B). The slices of in-gel tryptic digestion of mitotic condensin were subjected to mass spectrometry, and peptides derived from the heteropentameric subunits were identified (the coverage of peptides was 44–75%, supplementary material Fig. S2A). We detected a total of 18 phosphorylation sites in mitotic condensin (five, two, zero, nine and two sites in the Cut3, Cut14, Cnd1, Cnd2 and Cnd3 subunits, respectively). Both SMC and nonSMC subunits were thus phosphorylated. Because the coverage of Cnd1 was only 44%, it remains to be determined whether Cnd1 was actually phosphorylated. The locations of the phosphorylated peptides in the amino acid sequences are shown in Fig. 5C for the Barren-like Cnd2 subunit and in supplementary material Fig. S2B– D for the other three subunits. Consistent with a previous report (Sutani et al., 1999), the Cut3 T19 residue, in the Cdc2 consensus site [T19]PDR, was phosphorylated.

Identification of putative Aurora-B-like Ark1 sites in the Cnd2 subunit

We focused on the putative phosphorylation sites of the Aurora-Blike kinase Ark1 of S. pombe, as the nuclear chromosomal recruitment of condensin requires the Ark1-associating protein Bir1 (also known as Cut17) (Morishita et al., 2001; Nakazawa et al., 2008). The kinase-dead mutation Ark1-K118R prevents the localization of condensin to chromosomes and has a strong dominant-negative effect on mitotic condensin (Petersen and Hagan, 2003). The kinase Aurora B is essential for the chromosomal localization of condensin in Drosophila (Giet and Glover, 2001) and Caenorhabditis elegans (Hagstrom et al., 2002). In vertebrates, Aurora B is required for enrichment of condensin II at kinetochores (Ono et al., 2004) and also for facilitating the localization of condensin I at mitotic chromosomes (Lipp et al., 2007; Takemoto et al., 2007). The consensus sequence of Aurora kinase (R/K-xS/T-, where  indicates a hydrophobic residue) (Cheeseman et al., 2002) was used to search for the putative Aurora target sites. The

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five condensin subunits contained a total of 34 potential Aurora target sites, 23 of which were covered by the mass spectrometric analysis. Among these, three sites (S5, S41, S52) in the N-terminal region of the Barren-like Cnd2 subunit were indeed phosphorylated, as determined by mass spectrometry (Fig. 5C).


Preparation of a polyclonal antibody against a Cnd2 phosphorylated site

To detect phosphorylation of the Aurora kinase site, antibodies against the phosphopeptide [S45]ITPRRE-[S-P]-LNNS[S57], phosphorylated at S52, were raised (see Materials and Methods). The resulting antibodies were then used for immunoblotting of phosphorylated Cnd2 in S. pombe asynchronous (33°C, AS) and nda3-arrested (20°C, 9 hours, mitosis) extracts. As a negative control, we constructed S. pombe strains (cnd2-A or cnd2-E) in which the S52 codon was chromosomally replaced with alanine or glutamate codons (A and E) and tagged with GFP (see below). As shown in Fig. 6A, phosphorylated S52 was weakly detected in the asynchronously grown culture (at 33°C) but was more intense in mitotic extracts (20°C, 9 hours). However, phosphorylation was almost undetectable in extracts from the replacement mutants. Hence, we concluded that the phosphopeptide antibody was specific for S52-P, and that it intensely interacted with Cnd2 in nda3 mitotic extracts. The other synthetic phosphopeptides tested did not produce appropriate antibodies. Cnd2 S52 is phosphorylated throughout mitosis

To confirm that S52 phosphorylation peaked in mitosis, a block– release experiment was performed using the temperature-sensitive cdc25-22 mutant (Fig. 6B). These cells were blocked in late G2 phase and released synchronously into mitosis by a temperatureshift from the restrictive 36°C to the permissive 26°C temperature. The timing of late anaphase or telophase was monitored by the increase in bi-nucleated cells without the septum. The G1–S phase corresponded to the increase in the number of bi-nucleated cells with a septum. The band for phosphorylated S52 was negligible in late G2 (0 minutes) but became intense and reached maximum intensity at 15–45 minutes after the release to 26°C. The peak of S52 phosphorylation coincided with the period from early mitosis to late anaphase and telophase. A weak, but still substantial, phosphorylated S52 band was detected at ~60 minutes, at the time corresponding to the onset of cytokinesis. We concluded that S52 phosphorylation was high and sustained throughout mitosis. By contrast, phosphorylation of Cut3 T19 by Cdc2 kinase, as detected by specific antibodies (Sutani et al., 1999), only briefly peaked at 15 minutes, corresponding to prophase and metaphase (Fig. 6B). Inhibition of the analog-sensitive Ark1-as3 abolishes S52 phosphorylation

To determine whether phosphorylation of the Cnd2 S52 residue was dependent on Ark1, the antibody against S52-P was used to immunoblot mitotic extracts obtained from a strain with a chromosomal replacement of Ark1 with the ark1-as3 allele (Hauf et al., 2007). This allele encodes an analog-sensitive protein that is specifically inactivated with 1-tert-butyl-3-naphthalen-1-ylmethyl1H-pyrazolo[3,4-d]pyrimidin-4-ylemine (1NM-PP1) (Papa et al., 2003). The ark1-as3 mutant was crossed into the cdc25-22 Cnd28Myc strain. The resulting strain was then used for the Cdc25 block–release experiment using DMSO as the control (Fig. 6C, left-hand panels). To specifically inhibit Ark1 activity during mitosis, 5 mM 1NM-PP1 was added to the G2-arrested culture

Fig. 5. Mitotic phosphorylation sites in condensin subunits. (A)A schematic showing condensin subunits in S. pombe and the name for SMC subunits. Cnd2 belongs to the Barren family. (B)The silver-stained SDSPAGE pattern of mitotic condensin obtained by immunoprecipitation using antibodies against FLAG from extracts of arrested cold-sensitive nda3-KM311 cells carrying Cut14–FLAG (FLAG). Extracts from the cold-sensitive nda3KM311 strain without the FLAG tag (‘No tag’) were used as a control. (C)The mass spectrometric identification of phosphorylation sites in Cnd2 (red S with residue numbers) is shown. The amino acid sequence underlined with the dashed line indicates the phosphopeptide containing S52-P, which was synthesized and for which a rabbit antibody was successfully obtained. Light gray sequences indicate peptides that were not recovered by mass spectrometry. The boxed sequences represent predicted sites for the kinase Aurora B (Cheeseman et al., 2002). The locations of the nine identified phosphorylation sites are shown in the cartoon, along with the cnd2-1 mutation site.

(36°C) at 15 minutes before the release (26°C) into mitosis (Fig. 6C, right-hand panels). The S52-P residue of Cnd2 was detected during mitosis (from 30 to 90 minutes) in the presence of DMSO, whereas the S52-P band was not detected at all in the presence of 1NM-PP1. This result strongly suggests that Ark1 is responsible for the phosphorylation of the Cnd2 S52 residue. The addition of 1NM-PP1 increased the frequency of chromosome segregation defect displaying phi-shaped and streaked phenotypes, which also closely resembled the phenotype of the cut17-275 mutant and of an Ark1 shut-off strain, confirming that Ark1 activity was inhibited by 1NM-PP1 (Hauf et al., 2007; Morishita et al., 2001; Petersen and Hagan, 2003). Mitotic progression was then quantitatively monitored by staining for the two types of the mitotic spindle (short spindle, 4 mm) with an anti-TAT1 antibody (Fig. 6C, bottom panels). The modes of spindle dynamics during the synchronous mitosis were basically identical in the presence or absence of 1NM-PP1.


Ark1-dependent phosphorylation of Cnd2 is required for proper mitosis

To determine whether the three phosphorylated sites (S5, S41 and S52) are functionally relevant for Cnd2, we constructed multiple strains with replacements of these serine residues with alanine (A) and glutamate (E) as depicted in Fig. 7A. All integrated strains, except strains 3A and 2E-2, could grow and form colonies. The reason why integrants 3A and 2E-2 could not be obtained is unknown, but the 3A mutant is likely to be inviable (see below). Light microscopy of DAPI-stained A and E substitution mutants revealed substantial proportions of A mutant cells (but not of E mutants) with abnormal mitosis, particularly in late anaphase. The frequencies of abnormal anaphase were semi-quantitatively designated (Fig. 7A). Typical segregation abnormalities in different substitution mutant strains are shown in Fig. 7B.


Although Cnd2 S5 and S41 were phosphorylated, it was unknown whether phosphorylation was upregulated in mitosis and was diminished by 1NM-PP1. To clarify this issue, mass spectrometry was performed for Cnd2-3FLAG protein obtained from a cdc25-22 Cnd2-3FLAG ark1-as3 culture that was released into anaphase in the presence or the absence of 1NM-PP1. For S5, seven peptides were obtained in the presence of DMSO, and two of them were phosphorylated (data not shown). In the presence of 1NM-PP1, nine peptides were obtained but none of them was phosphorylated. For S41, only one of eight peptides was phosphorylated in DMSO, but none of the nine peptides detected with 1NM-PP1 was phosphorylated. These results are not conclusive but suggest that there is an Ark1-dependent partial phosphorylation of S5 and S41. The peptide containing S52 was not covered in this experiment.

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Fig. 6. Mitotically regulated phosphorylation of Cnd2 S52 by Ark1. (A)The specificity of polyclonal antibodies against the S52-P-containing phosphopeptide was examined. Asynchronous (33°C) and mitotic (20°C, 9 hours) extracts were prepared from cold-sensitive nda3-KM311 Cnd2–GFP cells and were subjected to SDS-PAGE. Binding specificity was examined by comparison with wild-type (WT) Cnd2 with the substitution mutants A and E carrying the non-phosphorylatable alanine or phosphorylation mimetic glutamate residues. The arrow indicates the position of the Cnd2 band. Ponceau staining was used for the loading control of extracts. The asterisks indicate non-specific contamination bands. (B)Results of the block–release experiment using the cdc25-22 Cnd2-8Myc strain. Cells were arrested at late G2 at 36°C and then released at 26°C. Both Cnd2 S52 and Cut3 T19 phosphorylation were detected (the arrows indicate band positions). The antiTAT1 antibody against tubulin was used as a loading control. (C)A block–release experiment using the cdc25-22 strain containing the two integrated markers, Cnd2-8Myc and the analog-sensitive ark1-as3 allele (see the text for details). Two cultures with the control DMSO or the inhibitor (5mM 1NM-PP1) were prepared. These compounds were added to the cultures 15 minutes before release into mitosis at 26°C. S52 phosphorylation was not detected in the culture treated with the inhibitor (top panel), which also showed aberrant segregation phenotypes displaying phi-shaped and streaked chromosomes (middle panel). Mitotic progression was quantitatively monitored by staining for the two types of the mitotic spindle (short spindle, 4mm) with anti-TAT1 antibody (bottom panel). AS, asynchronous culture.


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Fig. 7. cnd2 mutants that cannot be phosphorylated by Ark1 are defective in segregation. (A)Multiple alanine and glutamate residue (A and E, respectively) substitutions were chromosomally integrated in the cnd2 gene as indicated. The frequencies of abnormal anaphase were then analyzed. ++, 20–50%; +, 10–20%; –,